Recombinant live avian vaccine, using as vector the avian infectious laryngotracheitis virus

ABSTRACT

The recombinant live avian vaccine comprises as vector an ILTV virus comprising and expressing at least one heterologous nucleotide sequence, this nucleotide sequence being inserted into the insertion locus formed by the intergenic region located between the stop codons of ORF B and ORF C of ILTV, which region, in a particular strain of ILTV, is defined between nucleotides 908 and 994 in SEQ ID NO:1.

This application claims priority from French application number 96 15687, filed Dec. 16, 1996, incorporated herein by reference.

The present invention relates to vaccines for avian used based on infectious laryngotracheitis virus (ILTV), into which at least one heterologous nucleotide sequence, in particular coding for and expressing an antigenic polypeptide of an avian pathogenic agent, has been inserted by genetic recombination under conditions providing for an immunization leading to an effective protection of the vaccinated animal against the said pathogenic agent.

The infectious laryngotracheitis virus (ILTV) is an alphaherpes virus (B. Roizman, Arch. Virol. 1992. 123. 425-449) which causes a serious respiratory pathology (infectious laryngotracheitis or ILT) in chickens (L. E. Hanson and T. J. Bagust, Diseases of Poultry 9th edn 1991. pp 485-495. Ames, Iowa State University Press). The vaccines currently available against this illness contain an attenuated strain which can be administered via different routes, including the intranasal, conjunctival and cloacal routes, in the drinking water and by aerosol (L. E. Hanson and T. J. Bagust, Diseases of Poultry 9th Edition 1991. pp 485-495. Ames, Iowa State University Press).

Molecular biology studies of the ILTV virus have enabled the viral genome to be characterized (M. A. Johnson et al., Arch. Virol. 1991. 119. 181-198) and a few genes of the virus to be identified (A. M. Griffin, J. Gen. Virol. 1989. 70. 3085-3089), including the genes coding for thymidine kinase (UL23) (A. M. Griffin and M. E. G. Boursnell, J. Gen. Virol. 1990. 71. 841-850; C. L. Keeler et al., Avian Dis. 1991. 35. 920-929), the glycoprotein gB (UL27) (A. M. Griffin, J. Gen. Virol. 1991. 72. 393-398; K. Kongsuwan et al., Virology 1991. 184. 404-410; D. J. Poulsen et al., Virus Genes 1991. 5. 335-347), the glycoprotein gC (UL44) (D. H. Kingsley et al., Virology 1994. 203. 336-343), the p40 capsid protein (UL26) (A. M. Griffin, Nucl. Acids Res. 1990. 18. 3664), the protein homologous to the herpes simplex (HSV-1) protein ICP4 (M. A. Johnson et al., Virus Research 1995. 35. 193-204), the proteins homologous to the HSV-1 proteins ICP27 (UL54), glycoprotein gK (UL53) and DNA helicase (UL52) (M. A. Johnson et al., Arch. Virol. 1995. 140. 623-634), ribonucleotide reductase (A. M. Griffin, J. Gen. Virol. 1989. 70. 3085-3089, WO-A-90/02802), the genes UL1 to UL5 (W. Fuchs and T. C. Mettentleiter, J. Gen. Virol. 1996. 77. 2221-2229), and the genes present in the short unique sequence of the genome (U_(S)) (M. A. Johnson et al., DNA Sequence--The Journal of Sequencing and Mapping 1995. Vol. 5. pp 191-194; K. Kongsuwan et al., Arch. Virol. 1995. 140. 27-39; K. Kongsuwan et al., Virus Research 1993. 29. 125-140; K. Kongsuwan et al., Virus Gene 1993. 7. 297-303; M. A. Wild et al., Virus Genes 1996. 12. 107-116; WO-A-92/03554; WO-A-95/08622.

An objective of the present invention is to develop an avian vaccine based on recombinant ILTV virus expressing a heterologous gene, this virus being capable of replication and of including an immunity in the vaccinated host while retaining the property of being perfectly harmless.

Another objective of the invention is to provide such a vaccine which is, at the same time, especially effective against infectious laryngotracheitis (ILT).

Another objective of the invention is to provide such a vaccine which can be used in mass vaccination via the mucosal route, for example by means of an aerosol or in the drinking water, such that the replication of the virus in the mucosae enables a mucosal and systemic immunity to be induced. Such a Mucosal immunity will be especially effective for combating respiratory diseases, and also against other diseases for which the route of entry of the pathogenic agent is mucosal.

Another objective of the invention is to provide such a vaccine which can be used both in adults and in young animals.

A specific objective is to provide such a vaccine which can be used in mass vaccination via the mucosal route of very young animals such as one-day-old chicks.

Another objective of the invention is to provide a vaccine against ILT which has an enhanced efficacy relative to the parent strain and which might even possibly permit the insertion and expression of a heterologous gene.

In the course of their work on the ILTV virus, the inventors found a genomic region which proved entirely suitable as a site for insertion of heterologous genes. This made it possible to develop a recombinant live vaccine based on an ILTV vector into which is inserted at least one sequence coding for an avian immunogen, especially the protein HN and F of the Newcastle disease virus (NDV), and/or the glycoprotein gB of the Marek's disease virus (MDV), and/or the protein VP2 of the Gumboro disease virus (IBDV), and/or the proteins S and M of the infectious bronchitis virus (IBV). Such a vaccine incorporating a sequence coding NDV, MDV and/or IBV proteins provides for a satisfactory protection of the animals against Newcastle disease, against Marek's disease, against Gumboro disease and against infectious bronchitis, respectively.

Hence a subject of the present invention is a recombinant live avian vaccine comprising as vector the ILTV virus comprising at least one heterologous nucleotide sequence, in particular coding for and expressing an antigenic polypeptide of an avian pathogenic agent, inserted into the insertion locus formed by the intergenic region located between the stop codons of ORF B and ORF C of the ILTV virus, which region, in a particular ILTV strain, is defined between nucleotides 908 and 994 in the sequence SEQ ID NO:1.

While the particular sequence described in the application (SEQ ID NO:1) originates from the vaccinal strain of ILTV T-20 Dec. 8, 1966 (LT BLEN vaccine) obtained from select Laboratories (10026 Main Street P.O. Box 6, Berlin, Md. 21811, USA), it is quite obvious that a person skilled in the art will be able to use the other strains of ILTV, bearing in mind the information given in the present document regarding the vaccinal strain.

ORF B and ORF C correspond, respectively, to the UL3.5 and UL4 genes described in the paper by W. Fuchs and T. C. Mettentleiter (J. Gen. Virol. 1996. 77. 2221-2229) of a pathogenic strain obtained from D. Lutticken, Boxmeer, Holland. This paper in no way suggests that this intergenic region might be used as insertion locus.

The sequence referenced SEQ ID NO:19 reproduces, for this pathogenic strain, the sequence equivalent to SEQ ID NO:1. The intergenic region used as insertion locus in accordance with the invention is included in SEQ ID NO:19 between nucleotides 908 and 994.

Heterologous sequence is understood to mean a sequence which does not originate from this insertion locus, that is to say either a sequence whose source is not the ILTV virus or a sequence originating from another genomic region of this virus, or alternatively originating from another ILTV strain, in particular a virulent strain.

Insertion into the insertion region is understood, in particular, to mean simple insertion or insertion after total or partial deletion of the insertion locus.

It is possible to inert one or more expression cassettes each comprising at least one sequence to be expressed.

To express the inserted sequence, it is preferable to use a strong eukaryotic promoter such as the CMV immediate early (IE) promoter, the Rous sarcoma virus (RSV) LTR and the SV40 virus early promoter, CMV immediate early (IE) promoter is understood, in particular, to mean the fragment given in the examples as well as its subfragments that retain the same promoter activity. The CMV IE promoter can be the human promoter (HCMV IE) or the murine promoter (MCMV IE), or alternatively a CMV IE promoter of another origin, for example of monkey, rat, guinea pig or porcine origin.

Other promoters of viral or cellular origin may also be used.

Among promoters of viral origin, there may also be mentioned the promoters of genes of the ILTV virus (genes considered to be immediate early (ICP4, ICP27, etc.), early (thymidine kinase, DNA helicase, ribonucleotide reductase, etc.) or late (gB, gD, gC, gK, etc.)), of the Marek's disease virus (MDV), (gB, gC, pp38, pp14, ICP4, Meq, etc., genes) or of the herpes virus of turkeys (gB, gC, ICP4, etc., genes).

The nucleotide sequence inserted into the ILTV vector in order to be expressed can be any sequence coding for an antigenic polypeptide of an avian pathogenic agent, capable, when expressed under the favourable conditions brought about by the invention, of providing for an immunization leading to an effective protection of the vaccinated animal against the pathogenic agent. The nucleotide sequences coding for the antigens of interest for a given disease may hence be inserted under the conditions of the invention.

This nucleotide sequence inserted into the ILTV vector may also code for an immunomodulatory polypeptide, and in particular a cytokine.

As a noteworthy feature, the vaccines according to the invention may be used for the in ovo vaccination of one-day-old or older chicks and of adults. Different administration routes may be used: the parenteral route, or the mucosal routes such as the oronasal (drinking water, aerosol), conjunctival (drop in the eye) or cloacal route, with a preference for the routes permitting a mucosal mass vaccination (drinking water, aerosol).

The invention proves especially useful both for protection against respiratory pathologies and against systemic pathologies, by blocking the natural routes of entry of the pathogenic agent.

The invention may, in particular, be used for the insertion of a nucleotide sequence coding appropriately for an antigenic protein of the NDV virus, and especially the glycoprotein HN or the glycoprotein F. A recombinant live vaccine is thereby obtained affording, in addition to protection against infectious laryngotracheitis, a satisfactory protection against Newcastle disease.

The recombinant vaccine against Newcastle disease will preferably contain from 10 to 10⁴ pfu/dose.

Other preferred cases of the invention are the insertion of nucleotide sequences coding for antigens of other avian pathogenic agents, and in particular, but without implied limitation, antigens of the Marek's disease virus, especially gB, gC, gD and gH+gL genes (WO-A-90/02803), of the Gumboro disease virus, especially VP2 gene, of the infectious bronchitis virus (IBV), especially S and M genes (M. Binns et al., J. Gen. Virol. 1985. 66. 719-726; M. Boursnell et al., Virus Research 1984. 1. 303-313), of the chicken anaemia virus (CAV), especially VP1 (52 kDa)+VP2 (24 kDa) (N. H. M. Noteborn et al., J. Virol. 1991. 65. 3131-3139), of the ILTV virus, especially the genes coding for gB (A. M. Griffin, J. Gen. Virol. 1991. 72. 393-398), or for gD (M. A. Johnson et al., DNA Sequence--The Journal of Sequencing and Mapping 1995. Vol. 5. pp 191-194. Harwood Academic Publishers GmbH) or for gp60 (K. K. Kongsuwan et al., Virus Genes 1993. 7. 297-303) and of the virus of infectious swollen head syndrome (or chicken pneumovirosis or turkey rhinotracheitis virus (TRTV); pneumovirus), especially the fusion glycoprotein F (Q. Yu et al., J. Gen Virol. 1991. 72. 75-81) or the attachment glycoprotein G (R. Ling et al., J. Gen. Virol. 1991. 73. 1709-1715; K. Juhasz and J. Easton, J. Gen. Virol. 1994. 75 2873-2880). The doses were preferably the same as those for the Newcastle vaccine.

In the context of the present invention, it is naturally possible to insert more than one heterologous sequence into the said ILTV virus, in particular in this locus. It is possible, in particular, to insert therein sequences originating from the same virus or from different viruses, which also comprises the insertion of ILTV sequences and sequences from another avian virus. It is also possible to ally therewith sequences coding for immunomodulators, and especially cytokines.

For example, the CMV IE promoter is allied with another promoter so that their 5' ends are adjacent (implying transcriptions in opposite directions), which enables two nucleotide sequences, one under the control of the CMV IE promoter and the other under that of the allied promoter, to be inserted into the insertion zone. This construction is noteworthy for the fact that the presence of the CMV IE promoter, and in particular of its activating (enhancer) portion, activates the transcription induced by the allied promoter. The allied promoter can be, in particular, a promoter of a gene of the ILTV virus or of the MDV or HVT virus.

An advantageous case of the invention is a vaccine comprising a nucleotide sequence coding for NDV HN and a nucleotide sequence coding for NDV F or an antigen of another avian disease, in particular the ones mentioned above, one of the genes being under the control of the CMV IE promoter and the other under the control of the allied promoter.

It is also possible to introduce two CMV IE promoters of different origins with their 5' ends adjacent.

The expression of several heterologous genes inserted into the insertion locus may also be made possible by insertion between the open reading frames of these genes of a sequence known as "IRES" (internal ribosome entry site) originating, in particular, from a picornavirus such as the swine vesicular disease virus (SVDV; B. -F Chen et al., J. Virology, 1993, 67, 2142-2148), the encephalomyocarditis virus (EMCV; R. J. Kaufman et al., Nucleic Acids Research, 1991. 19, 4485-4490), the aphthous fever virus (FMDV; N. Luz and E. Beck. J. Virology, 1991, 65, 6486-6494), or alternatively from another origin. The content of the 3 papers cited is incorporated by reference. The cassette for expression of two genes would hence have the following minimum structure: promoter--gene 1--IRES--gene 2--polyadenylation signal. The recombinant live vaccine according to the invention may hence comprise, inserted into the insertion locus, an expression cassette comprising in succession a promoter, two or more genes separated pairwise by an IRES, and a polyadenylation signal.

In addition to the insertion into the locus according to the invention, it is possible to carry out one or more other insertions, one or more mutations or one or more deletions elsewhere in the genome; if the parent strain is virulent, it is possible, for example, to inactivate (by deletion, insertion or mutation) genes involved in the virulence, such as the thymidine kinase gene, the ribonucleotide reductase gene, the gE gene, etc. In any case, the insertion into a locus other than the one described in the invention enables other genes to be expressed.

A subject of the present invention is also a vaccine against ILT, comprising a recombinant ILTV virus into which an exogenous promoter, especially a strong promoter as described above, has been inserted upstream of the genes coding for major immunogens of ILTV, preferably the genes coding for gB (A. M. Griffin, J. Gen. Virol. 1991. 72, 393-398) or for gD (M. A. Johnson et al., DNA Sequence--The Journal of Sequencing and Mapping 1995. Vol. 5. pp 191-194. Harwood Academic Publishers GmbH) or for gp60 (K. K. Kongsuwan et al., Virus Genes 1993. 7. 297-303). This enables the level of expression of one or more of these genes to be increased, and thus makes it possible to produce a vaccine having enhanced efficacy against ILT. This may, of course, be combined with a construction as described above comprising the insertion of a heterologous sequence into the insertion locus.

A subject of the present invention is also a multivalent vaccine formula comprising, as a mixture or to be mixed, a vaccine as defined above with another vaccine, and in particular another recombinant live avian vaccine as defined above, these vaccines comprising different inserted sequences, in particular from different pathogens.

A subject of the present invention is also a method for preparing the vaccines according to the invention, as emerges from the description.

A subject of the present invention is also a method of avian vaccination, comprising the administration of a recombinant live vaccine or of a multivalent vaccine formula as defined above. Its subject is, in particular, such a method for the vaccination in ovo of one-day-old or older chicks and of adults. Different routes of administration of the vaccine may be used (see above), with a preference for the routes permitting a mass vaccination via the mucosal route (aerosol, drinking water), the dose of vaccine preferably being selected between 10¹ and 10⁴ per animal.

A subject of the present invention is also an ILTV virus comprising at least one heterologous nucleotide sequence as described above, inserted into the insertion locus as defined above.

A subject of the present invention is also all or part of the sequence SEQ ID NO:1; parts of this sequence are understood not only to mean the characterized ORFs taken separately or their fragments, but also the intergenic region located between ORF B and ORF C and the fragments located on each side of this intergenic region, which can, where appropriate, include a part of this intergenic region and which may serve as flanking arms for a homologous recombination, a technique which is, moreover, perfectly well known to a person skilled in the art. Generally speaking, but without implied limitation, the flanking arms can have from 100 to 1500 base pairs.

The invention will now be described in greater detail by means of the non-limiting examples of implementation, taken with reference to the drawing, wherein:

FIG. 1: Restriction map of the cloned fragment and position of the ORFs

FIG. 2: Sequence of 4161 bp and translation of ORFs A, B, C and D of the Select Laboratories vaccinal strain T-20

FIG. 3: Scheme for obtaining the plasmid pEL157

FIG. 4: Scheme for obtaining the plasmid pEL024

FIG. 5: Scheme for obtaining the plasmid pEL027

FIG. 6: Diagram of the plasmid pEL158

FIG. 7: Scheme for obtaining the plasmid pCD009

FIG. 8: Scheme for obtaining the plasmid pEL070

FIG. 9: Diagram of the plasmid pEL159

FIG. 10: Sequence of the NDV HN gene

FIG. 11: Scheme for obtaining the plasmid pEL030

FIG. 12: Diagram of the plasmid pEL160

FIG. 13: Diagram of the plasmid pEL033

FIG. 14: Diagram of the plasmid pEL161

FIG. 15: Diagram of double expression cassette

FIG. 16: Diagram of the plasmid pCD011

FIG. 17: Diagram of the plasmid pEL163

FIG. 18: Sequence of 4161 bp and translation of UL3, 3.5, 4 and 5 of Lutticken's pathogenic strain

SEQUENCE LISTING

SEQ ID NO:1 Sequence of the KpnI--KpnI fragment (4161 bp, see FIG. 2)

SEQ ID NO:2 Oligonucleotide EL001

SEQ ID NO:3 Oligonucleotide EL002

SEQ ID NO:4 Oligonucleotide EL003

SEQ ID NO:5 Oligonucleotide EL004

SEQ ID NO:6 Oligonucleotide MB070

SEQ ID NO:7 Oligonucleotide MB071

SEQ ID NO:8 Sequence of the NDV NH gene (see FIG. 10)

SEQ ID NO:9 Oligonucleotide EL071

SEQ ID NO:10 Oligonucleotide EL073

SEQ ID NO:11 Oligonucleotide EL074

SEQ ID NO:12 Oligonucleotide EL075

SEQ ID NO:13 Oligonucleotide EL076

SEQ ID NO:14 Oligonucleotide EL077

SEQ ID NO:15 Oligonucleotide CD001

SEQ ID NO:16 Oligonucleotide CD002

SEQ ID NO:17 Oligonucleotide CD003

SEQ ID NO:18 Oligonucleotide CD004

SEQ ID NO:19 Sequence of the KpnI--KpnI fragment (4161 bp, see FIG. 18)

EXAMPLES

All the plasmid constructions were carried out using the standard techniques of molecular biology described by Sambrook J et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. 1989). All the restriction fragments used for the present invention were isolated using the "Geneclean" kit (BIO101 Inc. La Jolla, Calif.).

The virus used as parent virus may be selected from the vaccinal strains described in J. R. Andreasen et al. (Avian Diseases 1990. 34. 646-656) or the strain T-20 Dec. 8, 1966 obtained from Select Laboratories 10026 Main Street P.O. Box 6 Berlin, Md. 21811, USA. It is also possible to use virulent strains such as Lutticken's strain (see above), the strain N-71851 (ATCC VR-783) or the strain 83-2 from USDA, which may be attenuated by known techniques, for example the one described in WO-A-95/08622.

Example 1

Culture of the ILTV virus:

The ILTV virus (Select Laboratories strain T20) is cultured on primary chicken kidney cells (CKC); these cells are cultured in MEM medium supplemented with 3% of foetal calf serum (FCS) in 75 cm² culture flasks (2×10⁵ cells/cm²) one or two days before inoculation.

On the day of inoculation, a flask of 1000 doses of lyophilized vaccine is resuspended in 10 ml of MEM medium supplemented with 1% of FCS; approximately 0.5 ml of this solution is then applied to the CKC culture. Next day, the medium is changed, and the day after, when the cytopathogenic effect (CPE) becomes generalized, the culture flasks are frozen at -70° C.

Culture of the ILTV virus may also be carried out on immortalized chicken liver cells, and in particular on the LMH line (W. M. Schnitzlein et al., Avian Diseases 1994. 38. 211-217).

Example 2

Preparation of ILTV genomic DNA:

After 2 cycles of freezing/thawing, the ILTV culture (two 75 cm² flasks) is harvested and centrifuged at low speed (5000 rpm in a 20 rotor, Beckman JA21 centrifuge, for 5 minutes) to remove large cell debris. The supernatant is ultracentrifuged (100,000 rpm, TLA100.3 rotor, Beckman TL100 centrifuge, for 1 hour). The pellet is then taken up in 1.6 ml of TEN-SDS (10 mM Tris pH 8.0; 1 mm EDTA; 0.5 NaCl; 0.5% sodium dodecyl sulphate), and 35 μl of a 20 mg/ml proteinase K solution are then added; the solution is incubated for 3 to 4 hours on a waterbath at 37° C., and the DNA is then extracted 3 times with phenol/chloroform and once with chloroform and is then precipitated with ethanol at -20° C. After centrifugation, the pellet is rinsed with 70% ethanol, dried and resuspended in 200 μl of TE (10 mM Tris pH 8.0; 1 mM EDTA). The nucleic acid concentration is then assayed in a spectrophotometer (OD₂₆₀). The DNA may be digested directly with the appropriate restriction enzymes and then cloned into the plasmid pBluescript II SK+; similarly, it may also be used in transfection experiments for obtaining a recombinant virus.

Example 3

Isolation and purification of recombinant ILTV virus:

The donor plasmid composed of a cassette for the expression of a polypeptide inserted between two flanking regions of the insertion locus is digested with a restriction enzyme permitting linearization of the plasmid, and it is then extracted with a phenol/chloroform mixture, precipitation with absolute ethanol and taken up in sterile water. 24-Hour primary CKC cells are then transfected with the following mixture: 0.2 to 1 μg of linearized donor plasmid+2 to 5 μg of ILTV viral DNA (prepared as in Example 2) in 300 μl of OptiMEM medium (Gibco BRI Cat # 041-01985H) and 100 μg of LipofectAMINE diluted in 300 μl of medium (final volume of the mixture=600 μl. These 600 μl are then diluted in 3 ml (final volume) of medium and plated out on 5×10⁶ CKC. The mixture is left in contact with the cells for 5 hours, then removed and replaced by 5 ml of culture medium. The cells are then left in culture for 3 to 8 days at +37° C. and thereafter, when the cytopathogenic effect has become apparent, they are frozen at -70° C. After thawing and, where appropriate, sonication, this viral population is cloned at limiting dilution in microplates (96-well) in order to isolate a homogeneous population of recombinant virus. These plates are left in culture for 1 to 3 days, and the supernatant is then harvested in an empty 96-well plate and the plate containing the supernatants is placed at 4° C. or at -70° C. The cells remaining in the other plates are then fixed with 95% acetone for 20 to 30 minutes at 20° C., or for 5 minutes at room temperature. An indirect immunofluorescence (IIF) reaction is carried out with a monoclonal antibody directed against the polypeptide expressed in order to look for plaques expressing this polypeptide. A further cloning is then performed in the same manner (at limiting dilution in 96-well plates) from the supernatant present in the wells of the plates placed at 4° C. or at -70° C. and corresponding to the wells displaying positive plaques in IIF. In general, 4 successive isolation cycles (limiting dilution, harvesting of the supernatant, cell monitoring by IIF, limiting dilution from the supernatant, etc.) suffice to obtain recombinant viruses all of whose progeny display a specific fluorescence. The genomic DNA of these recombinant viruses is characterized at molecular level by conventional PCR and Southern blot techniques using the appropriate oligonucleotides and DNA probes.

The isolation of recombinant virus may also be carried out by hybridization with a probe specific for the inserted expression cassette. For this purpose, the viral population harvested after transfection is diluted and applied to CKC cells (cultured in Petri dishes) so as to obtain isolated plaques. After 1 hour of contact at 37° C., the infection medium is removed and replaced by 5 ml of MEM medium containing 1% of agarose, kept supercooled at 42° C. When the agarose has solidified, the dishes are incubated for 48 to 72 hours at 37° C. in a CO₂ incubator until plaques have appeared. The agarose layer is then removed and a transfer of viral plaques is carried out on a sterile nitrocellulose membrane of the same diameter as the Petri dish used for culturing. This membrane is itself transferred onto another nitrocellulose membrane so as to obtain a reverse "copy" of the first transfer. The plaques transferred onto the latter copy are then hybridized according to the standard techniques known to the person skilled in the art with a digoxigenin-labelled DNA fragment (DNA Labelling Kit, Boehringer Mannheim, CAT # 1175033) of the expression cassette. After hybridization, washes and placing in contact with the visualization substrate, the nitrocellulose membrane is placed in contact with an autoradiographic film. The positive hybridization images on this membrane indicate which plaques are the ones containing recombinant ILTV viruses which have inserted the expression cassette. The plaques corresponding to these positive plaques are cut out under sterile conditions from the first nitrocellulose membrane, placed in an Eppendorf tube containing 0.5 ml of MEM medium and sonicated to release the virions from the membrane. The medium containing the Eppendorf tube is then diluted in MEM medium, and the dilutions thereby obtained are used to infect further cultures of CKC cells.

Example 4

Cloning and characterization of an ILTV genomic region:

The DNA extracted from the ILTV virus was digested with restriction enzyme KpnI for 2 hours at 37° C. The restriction enzyme was then removed by a phenol/chloroform extraction followed by an ethanol precipitation. The fragments resulting from this digestion were then ligated (overnight at 14° C.) with the plasmid pBluescript II Sk+ (pBS-SK+; Stratagene) digested with KpnI and treated with alkaline phosphatase; analysis of the clones obtained after transformation of E. coli DHA5α bacteria and culture on dishes of ampicillin-supplemented medium enables KpnI--KpnI inserts of different sizes to be identified, including a fragment of approximately 4.2 kb (plasmid pEL112).

Complete sequencing of the insert present in pEL112 (see FIG. 1) enabled two complete open reading frames (ORFs) (ORF B and ORF C) and a large part of two other ORFs (ORF A an ORF D) to be revealed. The restriction map of this cloned and sequenced genomic region is shown in FIG. 1; the sequence of 4161 bp (SEQ ID NO:1) is shown in FIG. 2. The position and the amino acid sequence of ORFs A, B, C and D are also shown in FIGS. 1 and 2, respectively.

The sequence between the stop codons of ORFs B and C (position from 908 to 994 on SEQ ID NO;1) can be used to insert cassettes for the expression of polypeptides into the ILTV genome. This sequence is referred to as the insertion locus. The insertion may take place with or without deletion in the intergenic region (see Example 5).

Example 5

Construction of the donor plasmid pEL157 for insertion into the intergenic region between ORFs B and C:

The plasmid pEL112 (7116 bp) was digested with the enzymes NotI and SpeI to isolate the 4.5-kb NotI-SpeI fragment. The fragment thus digested was then treated with the DNA polymerase (Klenow fragment) in the presence of dNTP in order to make the ends blunt; after ligation and transformation of E. coli bacteria, the clone pEL156 (4503 bp) was obtained.

The oligonucleotides EL001 (SEQ ID NO:2) and EL002 (SEQ ID NO:3) were used as primer for a first chain amplification by Taq polymerase (PCR). The oligonucleotides EL003 (SEQ ID NO:4) and EL004 (SEQ ID NO:5) were used as primer for a second chain amplification by Taq polymerase (PCR).

EL001 (SEQ ID NO:2): 5' TATTGCTTTCTACCGAAGTCGG 3'

EL002 (SEQ ID NO:3): 5' ACGCGAATTCAAATACGAGCATTTAATTATTGCG 3'

EL003 (SEQ ID NO:4): 5' TCTCCAGAATCGCTGGAGTGTCC 3'

EL004 (SEQ ID NO:5): 5' TGCGCGAATTCGTAAGCTTTGATATCCAGTCGACA TAATTTGGTGTTTATTACTTTTA 3'

The PCRs were performed in the presence of PCR buffer, dNTP, plasmid pEL156 DNA, Taq polymerase and, for the first PCR, the oligonucleotides EL001 and EL002, and for the second PCR, the oligonucleotides EL003 and EL004.

For both PCRs, 25 cycles were performed (30 seconds at 94° C.; 30 seconds at 60° C. and 30 seconds at 72° C.). The products of both PCRs were purified by a phenol/chloroform extraction followed by a purification with ethanol. The product of the first PCR (EL001/EL002) was then digested with the restriction enzymes XbaI and EcoRI for 2 h at 37° C. to give a 120-bp XbaI-EcoRI DNA fragment which was eluted after agarose gel electrophoresis. The product of the second PCR (EL003/EL004) was then digested with the restriction enzymes XhoI and EcoRI for 2 h at 37° C. to give an 85-bp XhoI-EcoRI DNA fragment which was eluted after agarose gel electrophoresis. Plasmid pEL156 was digested with the enzymes XbaI and XhoI. The two PCR fragments, XbaI and EcoRI (120 bp) and XhoI-EcoRI (85 bp), were ligated overnight at 14° C. with plasmid pEL156 digested with XbaI and XhoI. After transformation of E. coli bacteria and culture on dishes of ampicillin-supplemented medium, the clone pEL157 (4531 bp), comprising an EcoRI-HindIII-EcoRV-SalI polylinker, was obtained (see scheme for obtaining pEL157 in FIG. 3).

Example 6

Construction of the donor plasmid pEL158 for the insertion of a cassette for the expression of the IBDV VP2 gene under the control of the HCMV IE promoter into the intergenic site between ORFs B and C, and isolation of vILTV8:

6.1--Cloning of the Gumboro disease virus (IBDV) VP2 gene and construction of a cassette for the expression of VP2 under the control of the HCMV IE promoter

The plasmid pEL004 (see FIG. 4;=plasmid pGH004 described in French Patent Application 92/13109) containing the IBDV VP2 gene in the form of a BamHI-HindIII cassette was digested with BamHI and XbaI to isolate the 1104-bp BamHI-XBAI fragment (truncated VP2 gene). This fragment was cloned into the vector pBS-SK+ previously digested with XbaI and BamHI to give the plasmid pEL022 of 4052 bp (FIG. 4). The vector pBS-SK+ was digested with EcoRV and XbaI and then ligated with itself to give pBS-SK+ (modified). Plasmid pEL004 was digested with KpnI and HindIII to isolate the 1387-bp KpnI-HindIII fragment containing the complete IBDV VP2 gene. This fragment was cloned into the vector pBS-SK+ previously digested with KpnI and HindIII to give the plasmid pEL023 of 4292 bp (FIG. 4). Plasmid pEL022 was digested with BamHI and NotI to isolate the 1122-bp BamHI-NotI fragment (fragment A). Plasmid pEL023 was digested with BamHI and NotI to isolate the 333-bp BamHI-NotI fragment (fragment B). The fragments A and B were ligated together with the vector pBS-SK+ previously digested with NotI and treated with alkaline phosphatase to give plasmid pEL024 of 4369 bp (FIG. 4).

Plasmid pEL024 was digested with NotI to isolate the 1445 bp NotI-NotI fragment. This fragment was ligated with the plasmic pCMVβ (Clontech Cat # 6177-1, FIG. 5) previously digested with NotI to give the plasmid pEL026 of 5095 bp (FIG. 5).

Plasmid pEL026 was digested with EcoRI, SalI and XmnI to isolate the 2428 bp EcoRI-SalI fragment. This fragment was ligated with the vector pBS-SK+ previously digested with EcoRI and SalI to give plasmid pEL027 of 5379 bp (FIG. 5).

6.2--Construction of the donor plasmid pEL158

Plasmid pEL027 was digested with EcoRI, SalI and XmnI to isolate the 2428 bp EcoRI-SalI fragment. This fragment was ligated into plasmid pEL157 (see Example 5 and FIG. 3) previously digested with EcoRI an SalI to give plasmid pEL158 of 6950 bp (FIG. 6).

6.3--Isolation and purification of the recombinant virus vILTV8

The virus VILTV8 was isolated and purified after cotransfection of plasmid pEL158 DNA previously linearized with the enzyme KpnI and the viral DNA, as described in Example 3. This recombinant contains an HCMV-IE/IBDV VP2 cassette in the intergenic site between ORFs B and C of the ILTV virus (see Example 5).

Example 7

Construction of the donor plasmid pEL159 for the insertion of a cassette for the expression of the IBDV VP2 gene under the control of the MCMV IE promoter into the intergenic site between ORFs B an C, and isolation of vILTV9:

7.1--Construction of pEL070 containing a cassette for the expression of the IBDV VP2 gene under the control of the MCMV (mouse cytomegalovirus) immediate early (IE) promoter

Plasmid pCMVβ (Clontech Cat # 6177-1, FIG. 7) was digested with SalI and SmaI to isolate the 3679-bp SalI-SmaI fragment containing the lacZ gene as well as the polyadenylation signal of the SV40 virus late gene. This fragment was inserted into the vector pBS-SK+ previously digested with SalI and EcoRV to give the plasmid pCD002 of 6625 bp (FIG. 7). This plasmid contains the lacZ reporter gene, but no promoter is located upstream of this gene.

MCMV virus strain Smith was obtained from the American Type Culture Collection, Rockville, Md., USA (ATCC No. VR-194). This virus was cultured on Balb/C mouse embryo cells and the viral DNA of this virus was prepared as described by Ebeling A. et al. (J. Virol. 1983. 47. 421-433). This viral genomic DNA was digested with PstI to isolate the 2285-bp PstI--PstI fragment. This fragment was cloned into the vector pBS-SK+ previously digested with PstI and treated with alkaline phosphatase to give the plasmid pCD004 (FIG. 7). Plasmid pCD004 was digested with HpaI and PstI to isolate the 1389-bp HpaI-PstI fragment which contains the promoter/enhancer region of the murine cytomegalovirus (murine cytomegalovirus=MCMV) immediate early gene (Dorsch-Hasler K. et al. Proc. Natl. Acad. Sci. 1985. 82. 8325-8329 and Patent Application WO-A-87/03905. This fragment was cloned into plasmid pCD002 previously digested with PstI and SmaI to give plasmid pCD009 or 8007 bp (FIG. 7).

A double-stranded oligonucleotide was obtained by hybridization of the following two oligonucleotides:

MB070 (SEQ ID NO:6) 5' CGAATTCACTAGTGTGTGTCTGCAGGCGGCCGCGTGTGTGTCGACGGTAC 3'

MB071 (SEQ ID NO:7) 5' CGTCGACACACACGCGGCCGCCTGCAGACACACACTAGTGAATTCGAGCT 3'

This double-stranded oligonucleotide was ligated with the vector pBS-SK+ previously digested with KpnI and SacI to give the plasmid pEL067 (FIG. 8).

Plasmid pCD009 was digested with PstI and SpeI to isolate the 1396-bp PstI-SpeI fragment. This fragment was ligated with plasmid pEL067 previously digested with PstI and SpeI to give the plasmid pEL068 of 4297 bp (FIG. 8). Plasmid pEL024 (see Example 6, Section 6.1 and FIG. 5) was digested with HindIII and NotI to isolate the 1390-bp HindIII-NotI fragment (fragment A). Plasmid pEL027 (see Example 6, Section 6.1 FIG. 5) was digested with HindIII and SalI to isolate the 235-bp HindIII-SalI fragment (fragment B). The fragments A and B were ligated together with plasmid pEL068 previously digested with NotI and SalI to give plasmid pEL070 of 5908 bp (FIG. 8). This plasmid hence contains an expression cassette consisting of the MCMV IE promoter, the VP2 gene and the polyA signal of SV40).

7.2--Construction of the donor plasmid pEL159

Plasmid pEL070 was digested with EcoRI, SalI and XmnI to isolate the 3035-bp EcoRI and SalI fragment. This fragment was ligated into plasmid pEL157 (see Example 5 and FIG. 3) previously digested with EcoRI and SalI to give plasmid pEL159 of 7545 bp (FIG. 9). This plasmid permits the insertion of the MCMV IE/IBDV-VP2 expression cassette into the intergenic site between ORFs B and C of the ILTV virus.

7.3--Isolation and purification of the recombinant virus vILTV9

The virus vILTV9 was isolated and purified after cotransfection of plasmid pEL159 DNA previously linearized with the enzyme BglI and the viral DNA, as described in Example 3. This recombinant contains an MCMV-IE/IBDV VP2 cassette in the intergenic site between ORFs B and C of the ILTV virus (see Example 5).

Example 8

Construction of the donor plasmid pEL160 for the insertion of a cassette for the expression of the NDV HN gene into the intergenic site between ORFs B and C, and isolation of vILTV10:

8.1--Cloning of the Newcastle disease virus (NDV) HN gene

The building of a complementary DNA library for the genome of the Newcastle disease virus (NDV), strain Texas, was carried out as described by Taylor J. et al. (J. Virol. 1990. 64. 1441-1450). A pBR322 clone containing the end of the fusion gene (F), the whole of the haemagglutinin-neuraminidase (HN) gene and the beginning of the polymerase gene was identified as pHN01. The sequence of the NDV HN gene contained in this clone is presented in FIG. 10 (SEQ ID NO:8). The plasmid pHN01 was digested with SphI and XbaI to isolate the 2520-bp SphI-XbaI fragment. This fragment was ligated with the vector pUC19 previously digested with SphI and XbaI to give the plasmid pHN02 of 5192 bp. Plasmid pHN02 was digested with ClaI and PstI to isolate the 700-bp ClaI-PstI fragment (fragment A). A PCR was carried out with the following oligonucleotides:

EL071 (SEQ ID NO:9) 5' CAGACCAAGCTTCTTAAATCCC 3'

EL073 (SEQ ID NO:10) 5' GTATTCGGGACAATGC 3'

and the pHN02 template to produce a PCR fragment of 270 bp. This fragment was digested with HindIII and PstI to isolate a 220-bp HindIII-PstI fragment (fragment B). The fragments A and B were ligated together with the vector pBS-SK+ previously digested with ClaI and HindIII to give the plasmid pEL028 of 3872 bp (FIG. 11). Plasmid pHN02 was digested with BsphI and ClaI to isolate the 425-bp BsphI-ClaI fragment (fragment C). A PCR was carried out with the following oligonucleotides:

EL074 (SEQ ID NO:11) 5' GTGACATCACTAGCGTCATCC 3'

EL075 (SEQ ID NO:12) 5' CCGCATCATCAGCGGCCGCGATCGGTCATGGACAGT 3'

an the pHN02 template to produce a PCR fragment of 465 bp. This fragment was digested with BsphI and NotI to isolate the 390 bp BsphI-NotI fragment (fragment D). The fragments C and D were ligated together with the vector pBS-SK+ previously digested with ClaI and NotI to give the plasmid pEL029bis of 3727 bp (FIG. 11). Plasmid pEL028 was digested with ClaI and SacII to isolate the 960-bp ClaI-SacII fragment (fragment E). Plasmid pEL029-bis was digested with ClaI an NotI to isolate the 820-bp ClaI-NotI fragment (fragment F). The fragments E and F were listed together with the vector pBS-SK+ previously digested with NotI and SacII to give plasmid pEL030 of 4745 bp (FIG. 11).

8.2--Construction of plasmid pEL160 containing a cassette for the expression of NDV HN in the intergenic site between ORFs B and C

Plasmid pEL030 was digested with NotI to isolate the 1780 bp NotI--NotI fragment (complete NDV HN gene). This fragment was inserted into the NotI sites of plasmid pEL159 (Example 7, FIG. 9) in place of the 1405-bp NotI--NotI fragment containing the gene coding for the protein VP2 of IBDV; this cloning enables plasmid pEL160 of 7921 bp (FIG. 12) to be isolated. This plasmid permits the insertion of the MCMV-IE/NDV-HN expression cassette into the intergenic site between ORFs B and C of the ILTV virus.

8.3-13 Isolation and purification of the recombinant virus vILTV10

The virus vILTV10 was isolated and purified after cotransfection of plasmid pEL160 DNA previously linearized with the enzyme BglI and the viral DNA, as described in Example 3. This recombinant contains an MCMV-IE/NDV HN cassette in the intergenic site between ORFs B and C of the ILTV virus (see Example 5).

Example 9

Construction of the donor plasmid pEL161 for the insertion of a cassette for the expression of the NDV F gene into the intergenic site between ORFs B and C, and isolation of vILTV11:

9.1--Cloning of the Newcastle disease virus (NDV) F gene

A clone originating from the complementary DNA library for the Newcastle disease virus genome (see Example 8, Section 8.1) and containing the fusion gene (F) in its entirety was referred to as pNDV81. This plasmid has been described previously and the sequence of the NDV F gene present in this clone has been published (Taylor J. et al. J. Virol., 1990, 64. 1141-1450). Plasmid pNDV81 was digested with NarI and PstI to isolate the 1870-bp NarI-PstI fragment (fragment A). A PCR was carried out with the following oligonucleotides:

EL076 (SEQ ID NO:13) 5' TGACCCTGTCTGGGATGA 3'

EL077 (SEQ ID NO:14) 5' GGATCCCGGTCGACACATTGCGGCCGCAAGATGGGC 3'

and the pNDV81 template to produce a 160-bp fragment. This fragment was digested with PstI and SalI to isolate the 130-bp PstI-SalI fragment (fragment B). The fragments A and B were ligated together with the vector pBS-SK+ previously digested with CalI and SalI to give plasmid pEL033 of 4846 bp (FIG. 13).

9.2--Construction of plasmid pEL161 containing a cassette for the expression of the NDV F gene in the intergenic site between ORFs B and C

Plasmid pEL033 was digested with NotI to isolate the 1935-bp NotI--NotI fragment (complete F gene). This fragment was inserted into the NotI sites of plasmid pEL159 (Example 7, FIG. 9) in place of the 1405-bp NotI--NotI fragment containing the gene coding for the protein VP2 of IBDV; this cloning enabled plasmid pEL161 of 8074 bp (FIG. 14) to be isolated. This plasmid permits the insertion of the MCMV-IE/NDV-F expression cassette into the intergenic site between ORFs B and C of the ILTV virus.

9.3--Isolation and purification of the recombinant virus vILTV11

The virus vILTV11 was isolated and purified after cotransfection of plasmid pEL161 DNA previously linearized with the enzyme BglI and the viral DNA, as described in Example 3. This recombinant contains an MCMV-IE/NDV F cassette in the intergenic site between ORFs B and C of the ILTV virus (see Example 5).

Example 10

Construction of a donor plasmid for the insertion of a double cassette for the expression of the NDV HN and F genes into the intergenic site between ORFs B and C, and isolation of a recombinant ILTV virus:

A double cassette for the expression of two genes, for example the NDV HN and F genes, can be constructed. Such a construction is outlined in FIG. 15. In this construction, the 5' end of the two promoters are adjacent so the transcription of the two genes takes place in opposite directions. One of the two promoters is the MCMV IE promoter and the other promoter (referred to allied promoter) is the SV40 promoter (present in the plasmid pSVbeta, Clontech Laboratories, Palo Alto, Calif. 94303-4607, USA). In this arrangement, the allied promoter is activated by the enhancer region of the CMV IE promoter.

This double expression cassette may then be inserted into the donor plasmid described above (pEL157 described in Example 5 and shown in FIG. 3).

The isolation of the recombinant viruses takes place in the same manner as above (see Example 3).

Example 11

Construction of the donor plasmid pEL163 for the insertion of a cassette for the expression of the MDV gB gene into the intergenic site between ORFs B and C, and isolation of vILTV12:

11.1--Cloning of the gB gene of the Marek's disease virus

The 3.9-kbp EcoRI-SalI fragment of the genomic DNA of the MDV virus strain RB1B containing the MDV gB gene (sequence published by Ross N. et al. J. Gen. Virol. 1989. 70, 1789-1804) was ligated with the vector pUC13 previously digested with EcoRI and SalI to give the plasmid pCD007 of 6543 bp (FIG. 16). This plasmid was digested with SacI and XbaI to isolate the 2260-bp SacI-XbaI fragment (central part of the gB gene=fragment A). A PCR was carried out with the following oligonucleotides:

CD001 (D|SEQ ID NO:15) 5' GACTGGTACCGCGGCCGCATGCACTTTTTAGGCGGAATTG 3'

CD002 (SEQ ID NO:16) 5' TTCGGGACATTTTCGCGG 3'

and the pCD007 template to produce a PCR fragment of 222 bp. This fragment was digested with KpnI and XbaI to isolate a 190-bp KnpI-XbaI fragment (5' end of the gB gene=fragment B). Another PCR was carried out with the following oligonucleotides:

CD003 (SEQ ID NO:17) 5' TATATGGCGTTAGTCTCC 3'

CD004 (SEQ ID NO:18) 5' TTGCGAGCTCGCGGCCGCTTATTACACAGCATCATCTTCTG 3'

and the pCD007 template to produce a PCR fragment of 195 bp. This fragment was digested with SacI and SacII to isolate the 162-bp SacI-SacII fragment (3' end of the gB gene=fragment C). The fragments A, B and C were ligated together with the vector pBS-SK+ previously digested with KpnI and SacI to give plasmid pCD011 of 5485 bp (FIG. 16).

11.2--Construction of plasmid pEL163 containing a cassette for the expression of the MDV gB gene in the intergenic site between ORFs B and C of the ILTV virus.

Plasmid pCD011 was digested with NotI to isolate the 2608-bp NotI--NotI fragment (complete MDV gB gene). This fragment was inserted into the NotI sites of plasmid pEL159 (Example 7, FIG. 9) in place of the 1405-bp NotI--NotI fragment containing the gene coding for the protein VP2 of IBDV; this cloning enabled plasmid pEL163 of 8749 bp (FIG. 17) to be isolated. This plasmid permits the insertion of the MCMV-IE/MDV-gB expression cassette into the intergenic site between ORFs B and C of the ILTV virus.

11.3--Isolation and purification of the recombinant virus vILTV12

The virus vILTV12 was isolated and purified after cotransfection of plasmid pEL161 DNA previously linearized with the enzyme BglI and the viral DNA, as described in Example 3. This recombinant contains an MCMV-IE/MDV gB cassette in the intergenic site between ORFs B and C of the ILTV virus (see Example 5).

Example 12

Construction of a donor plasmid for the insertion of a cassette for the expression of IBV gene(s) into the intergenic site between ORFs B and C, and isolation of recombinant ILTV virus:

According to the same strategy as that described above for the insertion of single cassettes (Examples 6, 7, 8, 9 and 11) or for the insertion of double cassettes (Example 10) into the site described above (Example 5), it is possible to produce recombinant ILTV viruses expressing at a high level the membrane (M) or spike (S), or part of spike (S1 or S2) or nucleocapsid (N) proteins of the avian infectious bronchitis virus (IBV). In particular, a double expression cassette is produced with the S gene under the control of the CMV IE promoter and the M gene under the control of the allied promoter.

Example 13

Construction of donor plasmids for the insertion of cassettes for the expression of gene(s) of other avian pathogenic agents or of immunomodulatory peptide in the site described, and isolation of recombinant ILTV viruses:

According to the same strategy as that described above the insertion of single cassettes (Examples 6, 7, 8, 9 and 11) or for the insertion of double cassettes (Example 10) into the site described above (Example 5), it is possible to produce recombinant ILTV viruses expressing at a high level immunogens of CAV (and in particular a double cassette for the expression of the genes coding for VP1 and VP2), of the chicken pneumovirosis virus or of other avian pathogenic agents, or alternatively immunomodulatory peptides, and in particular cytokines.

Example 14

Production of vaccines:

The recombinant viruses obtained according to the invention are produced on embryonated eggs. The harvested viral solution is then diluted in a stabilizing solution for lyophilization, distributed on the basis of 1000 vaccinal does per vial and lastly lyophilized.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 19                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 4161 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Laryngotracheitis VirusInfectious                                             #Laboratories STRAIN: T20 Select                                               -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:1..585                                                            (D) OTHER INFORMATION:/par - #tial                                                  /product=- # "ORFA"                                             #"ORFA"        /gene=                                                          -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:689..904                                                          (D) OTHER INFORMATION:/pro - #duct= "ORFB"                           #"ORFB"        /gene=                                                          -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:complement (9 - #98..1531)                                        (D) OTHER INFORMATION:/pro - #duct= "ORFC"                           #"ORFC"        /gene=                                                          -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:complement (1 - #620..4160)                                       (D) OTHER INFORMATION:/par - #tial                                                  /product=- # "ORFD"                                             #"ORFD"        /gene=                                                          #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - GGTACCGAGA TCCCCTCTGT GACAGAAGTT CTATATGGAG CCTCGTCTAT TG - #ACTGTGTT          60                                                                           - TCCGGATCGT ACGAATGTCA TGATACGCAT TCTCATGTTA CTCCGGACCA CG - #CAAAAGAC         120                                                                           - GTAGCCGTCC AAGGGCATGT CAAAACGAAT AACACCGAAG ATGTAGAATC TT - #TGGACTCG         180                                                                           - TGTGGCTTTG ACAGTGTTTT CATGATATTT TCATTCGGGG AACTCGGGAG AA - #GACAGCTT         240                                                                           - ACCGATAACA TTCGAAAAGA CATTTGTACT TCCCTAGACA GAGTTCCGAT GG - #CATGTACT         300                                                                           - AAGACGTCCG CATTTGCAGG TGCAAATAGA CATCAGAAAA GTTTGCAGAT GT - #TTCTCTTT         360                                                                           - TGCAAGAGAA GACATGCCCC GCAAATAAGG GCTCGCCTAA AAGACATTAT TT - #CGTCAAGA         420                                                                           - AAGTCAAGAA AATATTTTAC GCAGTCCGAG GATGGAGAAA CTCACCCCGG TG - #TGCCAGTT         480                                                                           - TTCTTTCACG AGTTTGTAGC CCATGCCCCG GTATTTATTC CACGCGACAA TC - #TTGCCCAT         540                                                                           - GCCTGTCGCA GATTGGCCAG GCATATGACT GGAGGAATGG CGTGTTGACT TA - #CGTGGGCG         600                                                                           - CGCCTCTGGG TGGAACCCAG CGCTAGAACA TTTATACCTG CCCCATTGCG AA - #AGTTACTC         660                                                                           - AGAACGCGAA TATTGCACTT CCTGGACTAT GAAGTACGGT CAGGCTGCTA TT - #GCAACTGA         720                                                                           - TATGGACTTT GCCCGCATGT CTCGCCAGCC TCCACGGAAG AACTCCAGGT GC - #TCTTCGGC         780                                                                           - ACGGACGGCT GCGTTACAGG GTAATGGATA TTGCTTTCTA CCGAAGTCGG AA - #AAGTTGCC         840                                                                           - TGAGCTACCC TCTAGACATT TCGAGACCCG ATTTTCCTCA CTACTACCTC CA - #GCTGCAGC         900                                                                           - TAAGTGACAG CAACTATGAA GGTCAACTCA TTTCCGCAAT GCGACGCAAT AA - #TTAAATGC         960                                                                           - TCGTATTTCA TAATTTGGTG TTTATTACTT TTATTTATTC CTCTAACAGT CC - #GGCATGCC        1020                                                                           - TTGCCGCAAA CTCTACAAGA TCTCGAGGAA CGCTTTCCTC TGGACACTCC AG - #CGATTCTG        1080                                                                           - GAGAGGATTG GGAACATGTG GGGGTGGTGT GCGCGCTAGA TGCTAGATCT TC - #CGGGGTTT        1140                                                                           - CGTATATGGT TACAGTTAAG TGAGCGACGC CCAAAAAATT CATCATGGTG AT - #GTTGCCGC        1200                                                                           - TGCTCCACCG TTCTCGCGTT CCTCCGCGCC CTAGAAACCA ACATGCCGAG AA - #CTGAAAAG        1260                                                                           - CTAATGTTTC TCCTGAGGGT CCTCGGTGAG AACCATTGCG CTCCAAACAG TA - #TGCGCAAA        1320                                                                           - ATTCTTCTTC ACAGTCTACA GCGATCATTG TTGCGACGGG ATTGTGAATT AC - #TATTACTT        1380                                                                           - TCCCGGGTGG TAATTGGTGG CGATACATTT TATTTCCGAT GCAAATAAAC CG - #CATTCCTC        1440                                                                           - CATCGCATGA GTATACTACT TGTTCGTATT CGGTGGCCGA GCCATGCCCC AA - #GCCTTGGA        1500                                                                           - CTCCTACCAT TACGTAGCTT ATGAAAATCA TGTTCTCGGG ACGAGGAGGG TA - #TGCAACAC        1560                                                                           - TCCAAACGAC GGTGGGAAAC TGATTTTAAA GCCACGCGTG TGGCTACTTT TG - #AGGATTAG        1620                                                                           - TATACTAGTC GCGTTTCTTT GCATCTGAGC GCGCGAAGAA TGTGTTGGCT TA - #CTTGTCGA        1680                                                                           - GTATCTTCGT ATTTCGTTCG CAGGGGGTTT AAGTTCATGC GCAAATATTC GC - #TACTCGTT        1740                                                                           - ACTCTAGACA TTGCTACGTA TGCAGTATTT AATTTCATTG TTCCGTGAGA AA - #AACATATT        1800                                                                           - GCTACCCTGT CTAAACTTAA CCCTTGAGAT CGTGTGATCG TCATTGCAAG GC - #TCGAACTT        1860                                                                           - ATTCCGTGGT CAATTGTTAT GGCCATCCTT AATTCCTGTC CACCTATATT AT - #CGACAAAA        1920                                                                           - GTTGCTTTGT TATGGCACAG GACTGAAACG AACCCCATTT GATCCCTTAC GA - #CAAGTCTT        1980                                                                           - GGCAAGTCTA GCATTTGTAA AATTGGCTCG GCCCATTCGT GGGCTTTTGA GG - #TGTCCTCA        2040                                                                           - GCATATCCCG GAAATCTTGC TCGCGTAATC CCGCGTAAGG TATATGTGTC GG - #TTTGAGCA        2100                                                                           - GCAAATGCAC ATACTGCTCC CTTGAAACTG GAGATAGAAA TTTCTTGACT TG - #AGAAAGAA        2160                                                                           - GCCGTTCCGA CGAATGAGCT AAAGGGGGCG GCAGTAAATA CACTCCCAAA GA - #GCTCACAC        2220                                                                           - AGCACCGCGT AACGTAAAGT ATAAATTCTT TTCATCTGCT CGAAATAACT AA - #ATATTTCT        2280                                                                           - TGACCGTGGA CATCGCTCCC GCAAATCTCA TAATTTAGAT AGAATTGATC TA - #ACGACTTA        2340                                                                           - TCAAAAATAT CGAATAAATC ATCTTTGTCA TTCACATCGG CATTCGGACA TT - #CATCTTCG        2400                                                                           - TTAAAACAAA ATTTTTCACC ATTTCCCATG TCAAAGTTCT GAGTTTCTGG CT - #CCGGCATT        2460                                                                           - GCTAGGGAGT ACAACCTGTC ATACGCCATT GTTAATTTAT CTTCGGGGAG GC - #CCTCTGTT        2520                                                                           - CGGAGAAATT CGTAAAATTT AATCATCCCA TAGTATAGCA AGGTCGACAG AA - #AGTGATAG        2580                                                                           - GCAAATTCTA CTTTGTCTTC TCCATACGTC TTGAGAAAGC TATCTTCGGA TA - #ATACATGT        2640                                                                           - GCGAATTTTT CAAATGTTCC CTCAAAACCG AAGATCAATT TTTTTACCTT TT - #TTGTTACC        2700                                                                           - GTGATCTGGC TATTGAGTAC GTGCGAGGCA TCAGTCGTTA CTAGTGTATA TT - #CATTACTC        2760                                                                           - TCATCTCGAT GGTATTCAAA TCTGGGCGCG GTCACATCTA GATCCCTGCT CT - #GTGAATAG        2820                                                                           - TTTCCAAGGC GAGAGGAATT GTTTTGCAGC CAGCGATCAA TATTTAGTGT CT - #CTTGACCC        2880                                                                           - GTCAGGGATT TATACTTTTC AAATGCCGCC ATATCTACTA TTGTATACAT CG - #GCAGGAGA        2940                                                                           - AATACCCTAT ATTTTTCAGA TTTCTGTGCA CGAAGCTTTG CGTGCAATTT AG - #AAACGTAT        3000                                                                           - TCTTTAACTT CTTCGTGGGA CGAGAAAAGC CTAGTCCATC CAGGAAGCTT TG - #ATGGATCT        3060                                                                           - TTGATGAATG ATTCTGATAC AATGAACTGA TCTAAGAATC TGGCATGGCG TT - #TTGTCAAA        3120                                                                           - GGTAACCCAA ATTCAAATGC CTTTAATACT TCTCCGAAAG CAGGCTCCGA GC - #ATCGTTTA        3180                                                                           - TTATTAATAA AGATGGCCCA CTGCTTCTTG ATATTCAGGA CTGAAAACAG TG - #TAGGAGTA        3240                                                                           - CAAATAAGAT TACTTAAAAT GTTTATGCTG CTGGATATAA GGTGTCGTTG AT - #TTCTGTGC        3300                                                                           - TCGAAGCTGC TTTCCATTGC GTCTGTCTGT GTAGGGGAGC CAATGCACAC GA - #TCACTGGC        3360                                                                           - TTCTTCCCGT CCTGATACAT AGGCGTTTTC CACAATGCAT TCATGAGCCA CC - #ACGAATAT        3420                                                                           - ACTATTGCAG TCAATATATG TTTCCCTAAA ACTCCAGCCT CATCAACAAG GA - #TAATGTTG        3480                                                                           - CTTTTGACAA ATGGAGGCAT AGAGGAAATT AGGAAAGGTG CAAGGTTTAC AA - #ATTTGCGT        3540                                                                           - GAAGTTTTCT GCTGGAGTGT TTGAAGGACA GACAAAGCTA CCGGAGATGC CG - #TGTCTATG        3600                                                                           - GCGCGTGCAG TAATGTCCTT TATCACGTCC CAATAATAAT AAATGTCGGC CA - #TTTGATGT        3660                                                                           - TCTGCCAACG AACGTTGTTC GTGAGGTTTT TCAAACTTGA ATCGTCCTAG CA - #CAGCCTGT        3720                                                                           - ACGTTGTTTC CTTTGAAGCC AAAGTTTTGA AAAATAGTAT GAATGGGACA AG - #AGGTGTAA        3780                                                                           - GAGGCAGATA GCTTATTGAA GATATTAAGA GCAGCTATGC GCGTTGAGCC AG - #TAACGATG        3840                                                                           - CAATTCAATG TTTCATTAAG AGTTTGAATG CAAGTACTTT TTCCTGAGCC GG - #CGTTACCG        3900                                                                           - GTGATTAGAT AAACATTAAA TGGTAATTCC GCCAGAGGCA AAGTGGTCGG TT - #CATCTAAA        3960                                                                           - CGTGCCACAG TCTCAAACCA AGACAGTTGC GGCTTGGAGT CTTCATGAAC AG - #CTTGTTCT        4020                                                                           - GATAGGATTG TAATGTCCGA TAAAATCGCC TGAATGCTCT GCATTGCCGA AA - #AATTTAAG        4080                                                                           - TAAACAGGAG TGGTGATTTC TATTTCCCGC CTGCTCTTCC CCGAAAATGG AC - #ATGCCATT        4140                                                                           #                4161AC C                                                      - (2) INFORMATION FOR SEQ ID NO: 2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 22 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #laryngotracheitis virusInfectious                                             #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                 22GTC GG                                                     - (2) INFORMATION FOR SEQ ID NO: 3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 34 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #laryngotracheitis virusInfectious                                             #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #        34        GAGC ATTTAATTAT TGCG                                        - (2) INFORMATION FOR SEQ ID NO: 4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 23 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #laryngotracheitis virusInfectious                                             #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                23AGTG TCC                                                    - (2) INFORMATION FOR SEQ ID NO: 5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 58 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #laryngotracheitis virusInfectious                                             #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - TGCGCGAATT CGTAAGCTTT GATATCCAGT CGACATAATT TGGTGTTTAT TA - #CTTTTA            58                                                                           - (2) INFORMATION FOR SEQ ID NO: 6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 50 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #              50TGTGTC TGCAGGCGGC CGCGTGTGTG TCGACGGTAC                       - (2) INFORMATION FOR SEQ ID NO: 7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 50 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #              50CGGCCG CCTGCAGACA CACACTAGTG AATTCGAGCT                       - (2) INFORMATION FOR SEQ ID NO: 8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 2521 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #disease virusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION:303..2006                                                         (D) OTHER INFORMATION:/fun - #ction= "Hemagglutinin                                 Neuraminidas - #e"                                                             /product=- # "HN"                                               #"HN"          /gene=                                                                         /standard.sub.-- - #name= "HN"                                  #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - TGCTACCTGA TGTACAAGCA AAAGGCACAA CAAAAGACCT TGTTATGGCT TG - #GGAATAAT          60                                                                           - ACCCTTGATC AGATGAGAGC CACTACAAAA ATATGAATAC AAACGAGAGG CG - #GAGGTATC         120                                                                           - CCCAATAGCA ATTTGCGTGT AAATTCTGGC AACCTGTTAA TTAGAAGAAT TA - #AGAAAAAA         180                                                                           - CCACTGGATG TAAGTGACAA ACAAGCAATA CACGGGTAGA ACGGTCGGAG AA - #GCCACCCC         240                                                                           - TCAATCGGGA ATCAGGCCTC ACAACGTCCT TTCTACCGCA TCATCAATAG CA - #GACTTCGG         300                                                                           - TCATGGACCG TGCAGTTAGC AGAGTTGCGC TAGAGAATGA AGAAAGAGAA GC - #AAAGAATA         360                                                                           - CATGGCGCTT TGTATTCCGG ATTGCAATCT TACTTTTAAT AGTAACAACC TT - #AGCCATCT         420                                                                           - CTGCAACCGC CCTGGTATAT AGCATGGAGG CTAGCACGCC TGGCGACCTT GT - #TGGCATAC         480                                                                           - CGACTATGAT CTCTAAGGCA GAAGAAAAGA TTACATCTGC ACTCAGTTCT AA - #TCAAGATG         540                                                                           - TAGTAGATAG GATATATAAG CAGGTGGCCC TTGAGTCTCC ATTGGCGTTG CT - #AAACACTG         600                                                                           - AATCTGTAAT TATGAATGCA ATAACGTCTC TCTCTTATCA AATCAATGGA GC - #TGCAAATA         660                                                                           - ATAGCGGGTG TGGGGCACCT GTTCATGACC CAGATTATAT CGGGGGGATA GG - #CAAAGAAC         720                                                                           - TTATTGTGGA TGACGCTAGT GATGTCACAT CATTCTATCC CTCTGCGTTC CA - #AGAACACC         780                                                                           - TGAACTTTAT CCCGGCACCT ACTACAGGAT CAGGTTGCAC TCGGATACCC TC - #ATTCGACA         840                                                                           - TAAGCGCTAC CCACTACTGT TACACTCACA ATGTGATATT ATCTGGTTGC AG - #AGATCACT         900                                                                           - CACACTCATA TCAGTACTTA GCACTTGGCG TGCTTCGGAC ATCTGCAACA GG - #GAGGGTAT         960                                                                           - TCTTTTCTAC TCTGCGTTCC ATCAATTTGG ATGACAGCCA AAATCGGAAG TC - #TTGCAGTG        1020                                                                           - TGAGTGCAAC TCCCTTAGGT TGTGATATGC TGTGCTCTAA AATCACAGAG AC - #TGAGGAAG        1080                                                                           - AGGATTATAG TTCAATTACG CCTACATCGA TGGTGCACGG AAGGTTAGGG TT - #TGACGGTC        1140                                                                           - AATACCATGA GAAGGACTTA GACGTCATAA CTTTATTTAA GGATTGGGTG GC - #AAATTACC        1200                                                                           - CAGGAGTGGG GGGTGGGTCT TTTATTAACA ACCGCGTATG GTTCCCAGTC TA - #CGGAGGGC        1260                                                                           - TAAAACCCAA TTCGCCTAGT GACACCGCAC AAGAAGGGAG ATATGTAATA TA - #CAAGCGCT        1320                                                                           - ACAATGACAC ATGCCCAGAT GAACAAGATT ACCAGATTCG GATGGCTAAG TC - #TTCATATA        1380                                                                           - AGCCTGGGCG GTTTGGTGGA AAACGCGTAC AGCAGGCCAT CTTATCTATC AA - #GGTGTCAA        1440                                                                           - CATCTTTGGG CGAGGACCCG GTGCTGACTG TACCGCCTAA TACAATCACA CT - #CATGGGGG        1500                                                                           - CCGAAGGCAG AGTTCTCACA GTAGGGACAT CTCATTTCTT GTACCAGCGA GG - #GTCTTCAT        1560                                                                           - ACTTCTCTCC TGCTTTATTA TACCCTATGA CAGTCAACAA CAAAACGGCT AC - #TCTTCATA        1620                                                                           - GTCCTTACAC ATTCAATGCT TTCACTAGGC CAGGTAGTGT CCCTTGTCAG GC - #ATCAGCAA        1680                                                                           - GATGCCCCAA CTCATGTGTC ACTGGAGTTT ATACTGATCC GTATCCCTTA GT - #CTTCCATA        1740                                                                           - GGAACCATAC CTTGCGGGGG GTATTCGGGA CAATGCTTGA TGATGAACAA GC - #AAGACTTA        1800                                                                           - ACCCTGTATC TGCAGTATTT GATAACATAT CCCGCAGTCG CATAACCCGG GT - #AAGTTCAA        1860                                                                           - GCCGTACTAA GGCAGCATAC ACGACATCGA CATGTTTTAA AGTTGTCAAG AC - #CAATAAAA        1920                                                                           - CATATTGCCT CAGCATTGCA GAAATATCCA ATACCCTCTT CGGGGAATTC AG - #GATCGTTC        1980                                                                           - CTTTACTAGT TGAGATTCTC AAGGATGATG GGATTTAAGA AGCTTGGTCT GG - #CCAGTTGA        2040                                                                           - GTCAACTGCG AGAGGGTCGG AAAGATGACA TTGTGTCACC TTTTTTTTGT AA - #TGCCAAGG        2100                                                                           - ATCAAACTGG ATACCGGCGC GAGCCCGAAT CCTATGCTGC CAGTCAGCCA TA - #ATCAGATA        2160                                                                           - GTACTAATAT GATTAGTCTT AATCTTGTCG ATAGTAACTT GGTTAAGAAA AA - #ATATGAGT        2220                                                                           - GGTAGTGAGA TACACAGCTA AACAACTCAC GAGAGATAGC ACGGGTAGGA CA - #TGGCGAGC        2280                                                                           - TCCGGTCCCG AAAGGGCAGA GCATCAGATT ATCCTACCAG AGTCACATCT GT - #CCTCACCA        2340                                                                           - TTGGTCAAGC ACAAACTGCT CTATTACTGG AAATTAACTG GCGTACCGCT TC - #CTGACGAA        2400                                                                           - TGTGACTTCG ACCACCTCAT TATCAGCCGA CAATGGAAGA AAATACTTGA AT - #CGGCCACT        2460                                                                           - CCTGACACTG AGAGGATGAT AAAGCTCGGG CGGGCAGTAC ACCAGACTCT CG - #ACCACCGC        2520                                                                           #             2521                                                             - (2) INFORMATION FOR SEQ ID NO: 9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 22 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #                 22ATC CC                                                     - (2) INFORMATION FOR SEQ ID NO: 10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 16 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #    16                                                                        - (2) INFORMATION FOR SEQ ID NO: 11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 21 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #21                CATC C                                                      - (2) INFORMATION FOR SEQ ID NO: 12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 36 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #       36         CGCG ATCGGTCATG GACAGT                                      - (2) INFORMATION FOR SEQ ID NO: 13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 18 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #  18              GA                                                          - (2) INFORMATION FOR SEQ ID NO: 14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 36 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Newcastle                                                        (B) STRAIN: Texas GB                                                 #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #       36         ATTG CGGCCGCAAG ATGGGC                                      - (2) INFORMATION FOR SEQ ID NO: 15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 40 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Marek's                                                          (B) STRAIN: RB1B                                                     #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #    40            GCAT GCACTTTTTA GGCGGAATTG                                  - (2) INFORMATION FOR SEQ ID NO: 16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 18 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Marek's                                                          (B) STRAIN: RB1B                                                     #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #  18              GG                                                          - (2) INFORMATION FOR SEQ ID NO: 17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 18 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Marek's                                                          (B) STRAIN: RB1B                                                     #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #  18              CC                                                          - (2) INFORMATION FOR SEQ ID NO: 18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 41 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: YES                                                   -     (iv) ANTI-SENSE: YES                                                     -     (vi) ORIGINAL SOURCE:                                                    #Disease VirusORGANISM: Marek's                                                          (B) STRAIN: RB1B                                                     #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    #   41             GCTT ATTACACAGC ATCATCTTCT G                                - (2) INFORMATION FOR SEQ ID NO: 19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 4161 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: DNA (genomic)                                        -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                    #Laryngotracheitis VirusInfectious                                                       (B) STRAIN: Lutticken                                                #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - GGTACCTGAC GCAATTGGGA AAATGGCATG TCCATTTTCG GGGAAGAGCA GG - #CGGGAAAT          60                                                                           - AGAAATCACC ACTCCTGTTT ACTTAAATTT TTCGGCAATG CAGAGCATTC AG - #GCGATTTT         120                                                                           - ATCGGACATT ACAATCCTAT CAGAACAAGC TGTTCATGAA GACTCCAAGC CG - #CAACTGTC         180                                                                           - TTGGTTTGAG ACTGTGGCAC GTTTAGATGA ACCGACCACT TTGCCTCTGG CG - #GAATTACC         240                                                                           - ATTTAATGTT TATCTAATCA CCGGTAACGC CGGCTCAGGA AAAAGTACTT GC - #ATTCAAAC         300                                                                           - TCTTAATGAA ACATTGAATT GCATCGTTAC TGGCTCAACG CGCATAGCTG CT - #CTTAATAT         360                                                                           - CTTCAATAAG CTATCTGCCT CTTACACCTC TTGTCCCATT CATACTATTT TT - #CAAAACTT         420                                                                           - TGGCTTCAAA GGAAACAACG TACAGGCTGT GCTAGGACGA TTCAAGTTTG AA - #AAACCTCA         480                                                                           - CGAACAACGT TCGTTGGCAG AACATCAAAT GGCCGACATT TATTATTATT GG - #GACGTGAT         540                                                                           - AAAGGACATT ACTGCACGCG CCATAGACAC GGCATCTCCG GTAGCTTTGT CT - #GTCCTTCA         600                                                                           - AACACTCCAG CAGAAAACTT CACGCAAATT TGTAAACCTT GCACCTTTCC TA - #ATTTCCTC         660                                                                           - TATGCCTCCA TTTGTCAAAA GCAACATTAT CCTTGTTGAT GAGGCTGGAG TT - #TTAGGGAA         720                                                                           - ACATATATTG ACTGCAATAG TATATTCGTG GTGGCTCATG AATGCATTGT GG - #AAAACGCC         780                                                                           - TATGTATCAG GACGGGAAGA AGCCAGTGAT CGTGTGCATT GGCTCCCCTA CA - #CAGACAGA         840                                                                           - CGCAATGGAA AGCAGCTTCG AGCACAGAAA TCAACGACAC CTTATATCCA GC - #AGCATAAA         900                                                                           - CATTTTAAGT AATCTTATTT GTACTCCTAC ACTGTTTTCA GTCCTGAATA TC - #AAGAAGCA         960                                                                           - GTGGGCCATC TTTATTAATA ATAAACGATG CTCGGAGCCT GCTTTCGGAG AA - #GTATTAAA        1020                                                                           - GGCATTTGAA TTTGGGTTAC CTTTGACAGA ACGCCATGCC AGATTCTTAG AT - #CAGTTCAT        1080                                                                           - TGTATCAGAA TCATTCATCA AAGATCCATC AAAGCTTCCT GGATGGACTA GG - #CTTTTCTC        1140                                                                           - GTCCCACGAA GAAGTTAAAG AATACGTTTC TAAATTGCAC GCAAAGCTTC GT - #GCACAGAA        1200                                                                           - ATCTGAAAAA TATAGGGTAT TTCTCCTGCC GATGTATACA ATAGTAGATA TG - #GCGGCATT        1260                                                                           - TGAAAAGTAT AAATCCCTGA CGGGTCAAGA GACACTAAAT ATTGATCGCT GG - #CTGCAAAA        1320                                                                           - CAATTCCTCT CGCCTTGGAA ACTATTCACA GAGCAGGGAT CTAGATGTGA CC - #GCGCCCAG        1380                                                                           - ATTTGAATAC CATCGAGATG AGAGTAATGA ATATACACTA GTAACGACTG AT - #GCCTCGCA        1440                                                                           - CGTACTCAAT AGCCAGATCA CGGTAACAAA AAAGGTAAAA AAATTGATCT TC - #GGTTTTGA        1500                                                                           - GGGAACATTT GAAAAATTCG CACATGTATT ATCCGAAGAT AGCTTTCTCA AG - #ACGTATGG        1560                                                                           - AGAAGACAAA GTAGAATTTG CCTATCACTT TCTGTCGACC TTGCTATACT AT - #GGGATGAT        1620                                                                           - TAAATTTTAC GAATTTCTCC GAACAGAGGG CCTCCCCGAA GATAAATTAA CA - #ATGGCGTA        1680                                                                           - TGACAGGTTG TACTCCCTAG CAATGCCGGA GCCAGAAACT CAGAACTTTG AC - #ATGGGAAA        1740                                                                           - TGGTGAAAAA TTTTGTTTTA ACGAAGATGA ATGTCCGAAT GCCGATGTGA AT - #GACAAAGA        1800                                                                           - TGATTTATTC GATATTTTTG ATAAGTCGTT AGATCAATTC TATCTAAATT AT - #GAGATTTG        1860                                                                           - CGGGAGCGAT GTCCACGGTC AAGAAATATT TAGTTATTTC GAGCAGATGA AA - #AGAATTTA        1920                                                                           - TACTTTACGT TACGCGGTGC TGTGTGAGCT CTTTGGGAGT GTATTTACTG CC - #GCCCCCTT        1980                                                                           - TAGCTCATTC GTCGGAACGG CTTCTTTCTC AAGTCAAGAA ATTTCTATCT CC - #AGTTTCAA        2040                                                                           - GGGAGCAGTA TGTGCATTTG CTGCTCAAAC CGACACATAT ACCTTACGCG GG - #ATTACGCG        2100                                                                           - AGCAAGATTT CCGGGATATG CTGAGGACAC CTCAAAAGCC CACGAATGGG CC - #GAGCCAAT        2160                                                                           - TTTACAAATG CTAGACTTGC CAAGACTTGT CGTAAGGGAT CAAATGGGGT TC - #GTTTCAGT        2220                                                                           - CCTGTGCCAT AACAAAGCAA CTTTTGTCGA TAATATAGGT GGACAGGAAT TA - #AGGATGGC        2280                                                                           - CATAACAATT GACCACGGAA TAAGTTCGAG CCTTGCAATG ACGATCACAC GA - #TCTCAAGG        2340                                                                           - GTTAAGTTTA GACAGGGTAG CAATATGTTT TTCTCACGGA ACAATGAAAT TA - #AATACTGC        2400                                                                           - ATACGTAGCA ATGTCTAGAG TAACGAGTAG CGAATATTTG CGCATGAACT TA - #AACCCCCT        2460                                                                           - GCGAACGAAA TACGAAGATA CTCGACAAGT AAGCCAACAC ATTCTTCGCG CG - #CTCAGATG        2520                                                                           - CAAAGAAACG CGACTAGTAT ACTAATCCTC AAAAGTAGCC ACACGCGTGG CT - #TTAAAATC        2580                                                                           - AGTTTCCCAC CGTCGTTTGG AGTGTTGCAT ACCCTCCTCG TCCCGAGAAC AT - #GATTTTCA        2640                                                                           - TAAGCTACGT AATGGTAGGA GTCCAAGGCT TGGGGCATGG CTCGGCCACC GA - #ATACGAAC        2700                                                                           - AAGTAGTATA CTCATGCGAT GGAGGAATGC GGTTTATTTG CATCGGAAAT AA - #AATGTATC        2760                                                                           - GCCACCAATT ACCACCCGGG AAAGTAATAG TAATTCACAA TCCCGTCGCA AC - #AATGATCG        2820                                                                           - CTGTAGACTG TGAAGAAGAA TTTTGCGCAT ACTGTTTGGA GCGCAATGGT TC - #TCACCGAG        2880                                                                           - GACCCTCAGG AGAAACATTA GCTTTTCAGT TCTCGGCATG TTGGTTTCTA GG - #GCGCGGAG        2940                                                                           - GAACGCGAGA ACGGTGGAGC AGCGGCAACA TCACCATGAT GAATTTTTTG GG - #CGTCGCTC        3000                                                                           - ACTTAACTGT AACCATATAC GAAACCCCGG AAGATCTAGC ATCTAGCGCG CA - #CACCACCC        3060                                                                           - CCACATGTTC CCAATCCTCT CCAGAATCGC TGGAGTGTCC AGAGGAAAGC GT - #TCCTCGAG        3120                                                                           - ATCTTGTAGA GTTTGCGGCA AGGCATGCCG GACTGTTAGA GGAATAAATA AA - #AGTAATAA        3180                                                                           - ACACCAAATT ATGAAATACG AGCATTTAAT TATTGCGTCG CATTGCGGAA AT - #GAGTTGAC        3240                                                                           - CTTCATAGTT GCTGTCACTT AGCTGCAGCT GGAGGTAGTA GTGAGGAAAA TC - #GGGTCTCG        3300                                                                           - AAATGTCTAG AGGGTAGCTC AGGCAACTTT TCCGACTTCG GTAGAAAGCA AT - #ATCCATTA        3360                                                                           - CCCTGTAACG CAGCCGTCCG TGCCGAAGAG CACCTGGAGT TCTTCCGTGG AG - #GCTGGCGA        3420                                                                           - GACATGCGGG CAAAGTCCAT ATCAGTTGCA ATAGCAGCCT GACCGTACTT CA - #TAGTCCAG        3480                                                                           - GAAGTGCAAT ATTCGCGTTC TGAGTAACTT TCGCAATGGG GCAGGTATAA AT - #GTTCTAGC        3540                                                                           - GCTGGGTTCC ACCCAGAGGC GCGCCCACGT AAGTCAACAC GCCATTCCTC CA - #GTCATATG        3600                                                                           - CCTGGCCAAT CTGCGACAGG CATGGGCAAG ATTGTCGCGT GGAATAAATA CC - #GGGGCATG        3660                                                                           - GGCTACAAAC TCGTGAAAGA AAACTGGCAC ACCGGGGTGA GTTTCTCCAT CC - #TCGGACTG        3720                                                                           - CGTAAAATAT TTTCTTGACT TTCTTGACGA AATAATGTCT TTTAGGCGAG CC - #CTTATTTG        3780                                                                           - CGGGGCATGT CTTCTCTTGC AAAAGAGAAA CATCTGCAAA CTTTTCTGAT GT - #CTATTTGC        3840                                                                           - ACCTGCAAAT GCGGACGTCT TAGTACATGC CATCGGAACT CTGTCTAGGG AA - #GTACAAAT        3900                                                                           - GTCTTTTCGA ATGTTATCGG TAAGCTGTCT TCTCCCGAGT TCCCCGAATG AA - #AATATCAT        3960                                                                           - GAAAACACTG TCAAAGCCAC ACGAGTCCAA AGATTCTACA TCTTCGGTGT TA - #TTCGTTTT        4020                                                                           - GACATGCCCT TGGACGGCTA CGTCTTTTGC GTGGTCCGGA GTAACATGAG AA - #TGCGTATC        4080                                                                           - ATGACATTCG TACGATCCGG AAACACAGTC AATAGACGAG GCTCCATATA GA - #ACTTCTGT        4140                                                                           #                4161AC C                                                      __________________________________________________________________________ 

We claim:
 1. An ILTV virus comprising at least one heterologous nucleotide sequence inserted into the insertion locus formed by the intergenic region located between the stop codons of ORF B and ORF C of ILTV, which region, in a particular strain of ILTV, is defined between nucleotides 908 and 994 in SEQ ID NO:1.
 2. The ILTV virus of claim 1 wherein the at least one nucleotide sequence is inserted by simple insertion or after total or partial deletion of the insertion locus.
 3. The ILTV virus claim 1 including a strong eukaryotic promoter for expression of the at least one nucleotide sequence.
 4. The ILTV virus of claim 3 wherein the strong promoter is selected from the group consisting of: a CMV immediate early promoter, Rous sarcoma virus (RSV) LTR promoter, and SV40 virus early promoter.
 5. The ILTV virus of claim 4 wherein the CMV immediate early promoter is selected from the group consisting of the murine and the human CMV immediate early promoter.
 6. The ILTV virus of claim 1 comprising at least two nucleotide sequences inserted into the insertion locus under the control of different eukaryotic promoters.
 7. The ILTV virus of claim 6 wherein the eukaryotic promoters are CMV immediate early promoters of different animal origin.
 8. The ILTV virus of claim 6 comprising a first nucleotide sequence under the control of a first promoter and a second nucleotide sequence under the control of a second promoter, wherein the promoters are in the opposite orientation leading to transcription in opposite directions.
 9. The ILTV virus of claim 8 wherein the first promoter is the CMV immediate early promoter.
 10. The ILTV virus of claim 1 comprising an expression cassette inserted into the insertion locus, wherein the expression cassette comprises a promoter, two or more genes separated pairwise by an IRES, and a polyadenylation signal.
 11. The ILTV virus of claim 1 comprising a nucleotide sequence encoding an antigenic polypeptide of an avian pathogen inserted into the insertion locus.
 12. The ILTV virus of claim 11 wherein the nucleotide sequence encodes an antigenic polypeptide of Newcastle disease virus (NDV), the Gumboro disease virus (IBDV), the Marek's disease virus (MDV), the infectious bronchitis virus (IBV), the chicken anaemia virus (CAV), or the chicken pneumovirosis virus.
 13. The ILTV virus of claim 12 wherein the nucleotide sequence codes for NDV F or HN antigens.
 14. The ILTV virus of claim 12 wherein the nucleotide sequence codes for MDV gB, gC, gD or gH+gL antigens.
 15. The ILTV virus of claim 12 wherein the nucleotide sequence codes for at least one antigen selected from the group consisting of: IBDV VP2 antigen, IBV S, or part of S, M, and N antigens, CAV VP1 and VP2 antigens, and the chicken pneumonovirosis virus G and F antigens.
 16. The ILTV virus of claim 1 comprising a nucleotide sequence coding for an immunomodulatory polypeptide inserted into the insertion locus.
 17. The ILTV virus of claim 16 wherein the immunomodulatory polypeptide is a cytokine.
 18. An immunological composition comprising the ILTV virus of any one of claims 1-17.
 19. An immunological composition comprising as a mixture, or to be mixed, at least two ILTV viruses of any one of claims 1-17, wherein the ILTV viruses are comprised of different inserted sequences.
 20. A method for expressing at least one heterologous nucleotide sequence comprising contacting the ILTV virus of any one of claims 1-17 with a suitable cell under conditions for expression of the heterologous nucleotide sequence.
 21. Recombinant live avian vaccine comprising as vector an ILTV virus comprising and expressing at least one heterologous nucleotide sequence, this nucleotide sequence being inserted into the insertion locus formed by the intergenic region located between the stop codons of ORF B and ORF C of ILTV, which region, in a particular strain of ILTV, is defined between nucleotides 908 and 994 in SEQ ID NO:1.
 22. Recombinant live vaccine according to claim 21, characterized in that the nucleotide sequence or sequences is/are inserted by simple insertion or after total or partial deletion of the insertion locus.
 23. Recombinant live vaccine according to claim 21, characterized in that, to express the inserted nucleotide sequence, the vector comprises a strong eukaryotic promoter.
 24. Recombinant live vaccine according to claim 23, characterized in that the strong promoter is selected from the group consisting of: CMV immediate early promoter, Rous sarcoma virus (RSV) LTR promoter and SV40 virus early promoter.
 25. The recombinant live vaccine according to claim 24 wherein the CMV immediate early promoter is the murine or human CMV immediate early promoter.
 26. Recombinant live vaccine according to claim 21, characterized in that it comprises at least two nucleotide sequences inserted into the insertion locus under the control of different eukaryotic promoters.
 27. Recombinant live vaccine according to claim 26, characterized in that the eukaryotic promoters are CMV immediate early promoters of different animal origins.
 28. Recombinant live vaccine according to claim 26, characterized in that it comprises a first nucleotide sequence under the control of a first promoter and and a second nucleotide sequence under the control of a second promoter, wherein the promoters are in the opposite orientation leading to transcription in opposite directions.
 29. The recombinant live vaccine of claim 28 wherein the first promoter is the CMV immediate early promoter.
 30. Recombinant live vaccine according to claim 21, characterized in that it comprises, inserted into the insertion locus, an expression cassette comprising in succession a promoter, two or more genes separated pairwise by an IRES, and a polyadenylation signal.
 31. Recombinant live vaccine according to claim 21, characterized in that it comprises a nucleotide sequence coding for an antigenic polypeptide of an avian pathogenic agent, this sequence being inserted into the insertion locus.
 32. Recombinant live vaccine according to claim 31, characterized in that it comprises a sequence coding for an antigen of an avian pathogenic agent selected from the group consisting of the Newcastle disease virus (NDV), the Gumboro disease virus (IBDV), the Marek's disease virus (MDV), the infectious bronchitis virus (IBV), the chicken anaemia virus (CAV) and the chicken pneumovirosis virus.
 33. Recombinant live vaccine according to claim 32, characterized in that it comprises a nucleotide sequence selected from the nucleotide sequences coding for the polypeptides F and HN of the NDV virus.
 34. Recombinant live vaccine according to claim 32, characterized in that it comprises a nucleotide sequence selected from the nucleotide sequences coding for the polypeptides gB, gC, gD and gH+gL of the MDV virus.
 35. Recombinant live vaccine according to claim 32, characterized in that it comprises at least one nucleotide sequence selected from the group of sequences corresponding to the VP2 antigens of IBDV, to the S, or part of S, M and N antigens of the IBV virus, to the VP1 and VP2 antigens of CAV and to the G and F antigens of the chicken pneumovirosis virus.
 36. Recombinant live vaccine according to claim 21, characterized in that it comprises a nucleotide sequence coding for an immunomodulatory polypeptide, this sequence being inserted into the insertion locus.
 37. Recombinant live vaccine according to claim 36, characterized in that this nucleotide sequence is selected from the group of sequences coding for cytokines.
 38. Multivalent vaccine formula comprising, as a mixture or to be mixed, at least two recombinant live vaccines as defined in claim 21, these vaccines comprising different inserted sequences. 